ABSTRACT: Blood transfusion is the most common therapeutic procedure performed for hospitalized patients. Despite the efficacy of transfusion using standard donor-derived RBCs, we believe the full therapeutic potential of blood transfusion remains untapped. Recent work from several groups has demonstrated that human induced pluripotent stem cells (iPSCs) can be derived from small volumes of peripheral blood, can undergo genome editing to produce precise genetic changes, and can be differentiated into terminally mature RBCs (iPSC-RBCs), all under cGMP-compliant conditions. While iPSC-RBCs may not replace donor-derived RBCs for routine transfusions in the foreseeable future, we believe that iPSC-RBCs that have been genetically engineered to express novel functionalities (eg, prevention of alloimmunization in sickle cell disease patients) could find near term clinical applications in areas where current transfusion therapies are inadequate. In the proper therapeutic niche, engineered iPSC-RBCs would be high-value high-impact products that could sustain high costs due to their unique characteristics. The investigative team proposes to leverage recent advances in iPSC and genomic editing technologies, as well their ongoing NHLBI-funded studies in RBC biology and immunology, to pursue 3 Specific Aims: (1) To optimize cGMP-grade protocols for establishment, propagation, and differentiation of human peripheral blood-derived iPSCs into mature RBCs (iPSC-RBCs); (2A) To engineer iPSCs such that resulting RBCs are negative for multiple blood group antigens, and (2B) To characterize the in vivo survival, morphological maturation, and immunogenicity of iPSC-RBCs when transfused into specialized murine transfusion models; and (3) To obtain FDA IND and institutional IRB approvals, and perform human autologous transfusions of iPSC- RBCs to investigate RBC survival, maturation, and immunogenicity in recipients. The proposed studies have been carefully designed to integrate recently developed techniques (cGMP iPSC methods, gene editing) with specialized capabilities at Emory (EPIC cGMP clean room in the blood bank, in vivo biotinylated RBC tracking, first-in-human cell therapy expertise) to investigate the biology of human iPSC- derived RBCs after transfusion into human recipients. Successful completion of the proposed investigations will lead to novel blood products to meet important unmet clinical needs in transfusion-dependent patients such as those with sickle cell disease.